Sterile Alpha Motif (SAM) domains are among the most common protein modules in eukaryotic cells. They are found in many key regulatory proteins, scaffolding proteins, and transcription factors, which include many proteins involved in genetic diseases. Focused investigation of isolated systems has revealed that SAM domains are versatile protein-protein interaction partners, forming homo-oligomers, hetero-oligomers, homo- polymers, hetero-polymers and complexes with other macromolecules. Nevertheless, the vast majority of SAM domains remain functionally uncharacterized, leaving major gaps in our understanding of important biological pathways and disease states. Here we seek to leverage the considerable expertise we have developed with SAM domain structure and function to globally illuminate the functions of SAM domains in the human genome. The aims are: Aim I: Identify human SAM polymers. Every human-SAM domain will be expressed as a fusion to supercharged GFP to maintain polymer solubility. The SAM domains will be screened for aggregate formation by native gels and/or gel filtration chromatography. If they are found to form aggregates, they will be further examined by electron microscopy or AFM to distinguish aggregates from true polymers. Aim II: Identify all human homo- and hetero-SAM interactions discover polymer regulators and new copolymers. We will use a split DHFR assay to identify SAMs that bind to one another. Identified interactions will be verified biochemically with our GFP fusions from Aim I. We also hope to discover new SAM polymer regulators and possibly SAM copolymers. We believe the discovery-oriented approach proposed here is an efficient way to maximize the utility of our prior NIH funded advances for the benefit of others. PUBLIC HEALTH RELEVANCE: Cellular functions require precise communication between proteins. Disease occurs when these exchanges go awry. This project seeks to identify and characterize the interactions of a protein binding module found in about a hundred human proteins, including proteins directly involved in autism, leukemia, cleft palate, immune disorders, deafness and blindness. Thus, this project could illuminate the mechanisms of diverse disease states.